The overall objectives of the research projects described in this proposal are to provide insights into how myosin light chain kinase (MLCK) is regulated by Ca2+ / calmodulin in vivo and in vitro and to establish the importance of kinase binding to actin-containing filaments in smooth muscle. Specific aim I will test the hypothesis that Ca2+ / calmodulin activation of MLCK involves sequential binding steps between the two domains of calmodulin with the calmodulin-binding sequence and catalytic core. Low-angle X-ray and neutron-scattering studies will be combined with protein fragment complementation and protein cross-linking to identify sites of interactions between the catalytic core, the regulatory segment, and calmodulin. Specific aim II will determine the temporal and spatial distributions of calmodulin binding to MLCK in vivo with a biosensor MLCK containing fluorescent indicator proteins. The relationship between calmodulin-bound kinase and the extent of myosin regulatory light chain phosphorylation will be established. A biosensor MLCK will be expressed in transgenic mice with a smooth muscle-specific promoter for physiological studies on aortic and bladder tissues. Specific aim III will determine the biochemical mechanism for MLCK binding to actin-containing filaments. We will test the hypothesis that all three motifs cooperatively confer high-affinity binding and that spacing between the motifs is important. Specific aim IV will investigate the importance of MLCK binding to actin-containing filaments in vivo. We will test the hypothesis that the bound kinase is not translocated from F-actin filaments to the cytosol with increases in [Ca2+] in smooth muscle cells in culture and in tissues. Smooth muscle tissues play important roles in many body functions and are crucial for maintaining the homeostatic environment. The investigations proposed in this application address fundamental mechanisms involved in contractile regulation of smooth muscle. Investigations dealing with the primary biochemical pathway controlling smooth muscle contractility are essential for understanding derangements in smooth muscle-based diseases such as asthma, hypertension, erectile dysfunction and irritable bowl syndrome.